Ftf Gene Research Articles

Cariogenicity of Streptococcus mutans V403 glucosyltransferase and fructosyltransferase mutants constructed by allelic exchange

Streptococcus mutans produces several enzymes which metabolize sucrose. Three glucosyltransferase genes (gtfB, gtfC, and gtfD) and a single fructosyltransferase gene (ftf) encode enzymes which are important in formation of exopolysaccharides. Mutants of S. mutans V403 carrying single and multiple mutations of the gtfB, gtfC, gtfD, and ftf genes recently have been constructed by allelic exchange in our laboratory. Using selected strains from this panel of mutants, we examined the importance of water-insoluble glucan, water-soluble glucan, and fructan production in cariogenicity while controlling for the effects of strain and species variability. Genetic and biochemical characterization of mutants and assays of glucosyltransferase and fructosyltransferase activities were performed to ensure that the phenotypes of strains coincided with deficiencies predicted by genotype. The young gnotobiotic rat model of cariogenicity was used to assess virulence of the wild-type strain and isogenic mutants. Mutant strains were less virulent than the wild type in almost every location examined for caries on tooth surfaces and level of involvement of lesions (depth and severity). Inactivation of either gtfB and gtfC or ftf dramatically reduced virulence; the subsequent inactivation of gtfD did not enhance the effect of reduced virulence.

Biochemical characterization and evaluation of virulence of a fructosyltransferase-deficient mutant of Streptococcus mutans V403.

The Streptococcus mutans extracellular fructosyltransferase (FTF) enzyme may play a role in the formation of dental caries by synthesizing a fructan polymer that serves as an extracellular storage polysaccharide. We sought to determine if an FTF-deficient strain of S. mutans was less virulent than wild-type cells in a rat animal model system. Cloned ftf gene sequences from S. mutans GS5 were used to generate a defective copy of the ftf gene by inserting into the ftf coding region a DNA fragment which encoded erythromycin resistance. The plasmid which carried the defective ftf construct was introduced into S. mutans V403 by using genetic transformation. This defective construct replaced, by allelic exchange, the wild-type copy of the ftf gene carried on the V403 chromosome. FTF activity assays indicated that the recombinant strain, V1741, was deficient in fructan synthesis. However, extracellular protein preparations from this strain displayed an increased ability to generate glucose polymers (glucans) compared with V403 preparations. Levels of adherence to glass and rat tooth surfaces by strain V1741 were similar to those of the V403 strain. Both strains caused moderate decay on rat tooth surfaces; however, the FTF-deficient strain was less pathogenic compared with the wild-type strain. These results suggest that FTF activity contributes to the pathogenicity of S. mutans V403, possibly by generating extracellular fructans which serve as storage compounds.

Genetic and antigenic comparison ofStreptococcus mutansfructosyltransferase and glucan-binding protein

The genes for fructosyltransferase (ftf) and glucan-binding protein (gbp) of Streptococcus mutans strain Ingbritt have been cloned in E. coli using bacteriophage and plasmid vectors. A single ftf gene appears to be responsible for the appearance of several FTF of different electrophoretic mobilities. Despite the fact that both ftf and gbp proteins synthesise fructans from sucrose in S. mutans and shown closely similar electrophoretic mobilities and isoelectric points, the restriction maps of the two genes are distinct, their DNAs do not hybridise and there is no immunological cross-reaction between the two proteins.